Mycobacterium tuberculosis infections kill more than one million people annually. Although tuberculosis generally remains confined to the lung, mycobacteria also are able to disseminate to other tissues including bone. The interplay of bacterial and host factors that contribute to infection site, particularly bone infection, is incompletely understood. We have identified an outbreak strain that we find clinically to present with high rates of extrapulmonary dissemination and an unprecedented rate of tuberculous bone disease. We are analyzing this unique isolate of Mycobacterium tuberculosis to determine what strain- specific factors may be responsible for the proclivity to cause bone infection. We have sequenced and assembled the NCG genome and find that is a member of an ancient clade. We hypothesize that unique genetic variants of the NCG strain confer bone tropism. In order to understand the interactions of virulent mycobacteria with bone, we will 1) develop a zebrafish model to directly examine dissemination and bone disease during mycobacterial infection. Bone tissue is continuously remodeled by bone-forming osteoblasts and bone-resorbing osteoclasts; an imbalance in either or both processes can result in bone disease. Using a zebrafish model, we will observe in whole live animals in real time the impact of mycobacterial infection on bone tissue remodeling. 2) We will identify unique variants in the outbreak strain genome and use the zebrafish platform to assess their functional relevance in bone disease. 3) In a parallel approach, we will use mouse models to interrogate and validate specific genetic variants using aerosol infections with the outbreak strain. These studies will provide insights into the functiona significance of specific genetic variants during infection with an outbreak strain as well as the basis of mycobacterial dissemination and bone disease.